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What are E/K Coils?

E/K coils are synthetic coiled-coil domains designed specifically to bind to each other with high affinity and specificity (Litowski and Hodges, 2002) (Figure 1). They are composed of a heptad repeat that forms a coil structures that are able to interact with each other. These coils are able to interact with each other in an anti-parallel fashion that makes them useful for applications such as peptide capture, protein purification and in biosensors. For our project we decided to make use of the IAAL E3/K3 coils due to the balance they offer between affinity and specificity (Table 1).

How do these Coils Work?

These E3/K3 coils are able to form heterodimers due to the hydrophobic residues contained within the heptad repeat. In our case these are isoleucine and leucine residues. Designated by empty arrows in the helical wheel diagram below (Figure 2) these residues form the core of the binding domain of the coils. In order to prevent the homodimerization of these coils charged residues are included in the design. The electrostatic interactions between glutamic acid and lysine residues prevent an E-coil from binding with an E-coil for example. We selected the use of E3/K3 coiled-coils over other synthetic E/K coils as the isoleucine residue present shows a significant increase in the heterodimer over valine found in other coils. The alpha-helical propensity of the residues outside of the core interacting residues is also increased by utilizing an alanine residue instead of the serine residue found in ISAL and VSAL E/K coils. This selection maximizes the stability and specificity of the coils used in our system.

Ferritin Scaffold

One of the previously mentioned coils, the K-coil, will be attached to ferritin a ubiquitous 24-subunit, iron storage protein. Ferritin is typically composed of two subunits, called the heavy and light chains, although it has been previously shown that fusion of the two subunits will still result in a fully functionally nanoparticle. Therefore depending on how we construct our ferritin, we can create a scaffold that contains 24 or 12 K-coils. The fused ferritin subunits allow us to scale down our reporters, by reducing the number of attached reporters from 24 to 12. We can further scale the reporter down through the use of ferritin itself as a reporter, thus scaling the number of reporters down to one. This unique property of our system means it can be used in a wide range of areas, ranging from low to high sensitivity applications.

Another aspect of our Kcoil-ferritin system is the ability for us to interchange the proteins bound to the scaffold. We specifically used our DNA binding proteins, TALs, to target a sequence in pathogenic E.coli. But this system could easily be interchanged to target sequences in other organisms, by replacing our original TALs with an alternative TAL we can target any desired sequence of DNA. The ability to swap out proteins also means our system is not limited to targeting DNA sequences, but can be expanded towards other targets, such as proteins, with the only limitation being the number of known binding proteins.